白癜风患者血清自身抗体IgG识别人黑素浓集素受体-1B细胞表位的研究

B Cell Epitopes of Human Melanin-Concentrating Hormone Receptor 1 Recognized by Serum Autoantibodies from Patients with Vitiligo

目的寻找白癜风患者血清中抗体识别的人黑素浓集素受体-1(MCHR1)B细胞表位肽段.方法从重组质粒pcDNA3/MCHR1中PCR扩增包含预测抗原表位MCHR1肽段的基因,构建重组表达载体pGEX-4T/MCHR1,经限制性内切酶双酶切及DNA测序鉴定,转化大肠杆菌DH5α,利用谷胱甘肽巯基转移酶(GST)纯化试剂盒纯化表达的融合蛋白.采用酶联免疫吸附试验(ELISA)鉴定所表达的蛋白是否具有白癜风患者血清识别的表位.结果选择表达最可能包含B细胞表位的MCHR1氨基酸1～94(aa1～94),构建表达载体pGEX4T/MCHR1,得到纯化的重组蛋白GST-MCHR1相对分子质量为37 000.ELISA法检测结果显示,13.2%的白癜风患者血清抗MCHR1自身抗体IgG呈阳性反应.结论MCHR1aa1～94包含了白癜风患者自身抗体IgG识别的B细胞表位,为研究白癜风的发病机制奠定基础.

Objective To detect the B cell epitopes of human MCHR1 recognized by serum autoantibodies in patients with vitiligo. Methods The DNA encoding antigen fragments of MCHR1 from amino acid (aa) 1 to 94 were amplified from pcDNA3/MCHR1 by PCR and inserted into the vector pGEX-4T-1. After identification, the recombinant plasmid pGEX-4T-1/MCHR1 was transfected into E. coli for fusion protein production which was purified by GST agarose affinity chromatography later. The sera from 38 vitiligo patients were examined for MCHR1 antibodies by enzyme-linked immunoabsorbent assay. Results Amino acids (aa) 1 to 94 of MCHR1 which most possibly included in B cell epitopes were selected to express. The recombinant plasmid pGEX-4T /MCHR1 was confirmed by sequence analysis. SDS-PAGE showed that a Mr 37 kD fusion protein was expressed by E.coli. Of the sera from vitiligo patients examined by ELISA, 5 of 38 were considered positive for MCHR1 antibody. Conclusion There are B cell epitopes in amino acids(aa) 1 to 94 of MCHR1 which can be recognized by antibodies from vitiligo patients.